Effects of altering a single key amino acid on oxime-mediated reactivation in human and guinea pig acetylcholinesterases Alyssa Chalmin Mentored by Dr. Douglas Cerasoli and Ms. C. Linn Cadieux Introduction Results Results (continued) Colors: (74r, 26g, 102b); (226r, 153g, 148b); (223r, 131g, 125b); (147r, 145g, 142b) Organophosphorous nerve agents inhibit acetylcholinesterase (AChE), an enzyme used to control the concentration of acetylcholine at cholinergic synapses . Excess acetylcholine at these synapses causes neurological damage and, without intervention, leads to seizures, cardiorespiratory disruption, and death. (Ballantyne & Marrs, 1992). Oxime reactivators, a treatment for nerve agent exposure, remove the nerve agent from AChE which then resumes normal acetylcholine hydrolysis. Animal models provide a way for these treatments to be tested, and the more closely the model mirrors humans, the more predictive they are. Guinea pigs are a popular test subject because of their similarity to primates despite their classification as rodents. Guinea pig (GP) AChE and human (Hu) AChE react differently to select reactivators, so both were sequenced and compared (Cadieux et al., 2010). At amino acid residue 324, predicted to be near the binding site of the reactivators, GPAChE has an isoleucine, while HuAChE has a valine, shown in Figure 1. It is predicted that substituting isoleucine or valine at residue 324 will cause marked changes in oxime-assisted reactivation. of the enzyme to reactivate, was measured to be 0.75 min for wild type HuAChE and 3.61 min for mutant HuAChE. The F-test indicated significant difference with a p-value of 0.0002. The t1/2 for wild type GPAChE was measured to be approximately 5.4 x 1015 min, while the mutant GPAChE had a t1/2 of 72.3 min. The mutant reactivation rate had a significant difference from that of the wild type, shown by a p-value less than 0.0001. Hu AChE 361 VVKDEGSYFLVYGAPGFSKDNESLISRAEFLAGVRVGVPQVSDLAAEAVVLHYTDWLHPE GP AChE 360 ............................Q......I........................ Figure 1: This is a portion of the protein sequence comparison of human AChE and guinea pig AChE. The highlighted amino acid residue is residue 324, the site at which the substitution was made. 200 400 600 800 1,000 1 1,200 1,400 1,600 1,800 1,969 Figure 2: This image is the alignment of DNA sequences obtained through capillary laser sequencing. Primers were designed that together covered the full length of the sequence. The green bar shows consensus of the nucleotide bases among the different sequences. Conclusions The purpose of this project was to investigate the effects of amino acid substitutions on small-molecule mediated reactivation rates of nerve agent inhibited acetylcholinesterase. The mutant variant enzymes were successfully able to incorporate the amino acid substitutions into protein which expressed in mammalian human embryonic kidney cells and was active against AtCh. In HuAChE, substituting isoleucine for valine caused a marked (~5 fold) decrease in the reactivation potential of HI-6 against GB-inhibited enzyme (Graph 1). In GPAChE, substituting valine for isoleucine at residue 324 introduced reactivation of GB-inhibited enzyme when previously no reactivation could be detected in the wild type GPAChE (Graph 2). Together these findings demonstrate that amino acid residue 324 is important for the reactivation function of HI6 but is likely not the only amino acid contributing to reactivation potential in either enzyme. Future work includes mutating other amino acid residues that are close to the active site, such as residue 317 shown in Figure 1, full characterization of the mutated enzymes including full reactivation kinetics, and experiments with other reactivators and other agents. Graph 1: This graph shows the reactivation of GB-inhibited HuAChE after being exposed to HI-6. Reactivation is measured as the percent of the activity of inhibited enzyme without HI-6. The t1/2 is approximately five times lower in the mutated AChE than in the wild type AChE. Materials and Methods Mutated constructs of HuAChE and GPAChE were created by splicing the gene for AChE into pcDNA3.1(-) plasmids using BamH1 and HindIII restriction sites. The gene was codon-optimized for expression in mammalian cells. To confirm the sequences, primers were created through Integrated DNA Technologies, Inc. These primers were used in capillary laser sequencing using BigDye® terminator which contains dideoxynucleotides, allowing the entire sequence to be read as a progression of individual nucleotides. For each construct, the six sequences were aligned using Geneious, shown in Figure 2. After sequence confirmation, bacterial transformation of the constructs into DH5α E. coli bacteria was performed which, when incubated, caused DNA replication. A Qiagen Plasmid Maxiprep was then prepared to extract and purify the plasmid from the bacteria. The plasmid DNA was transfected into human embryonic kidney 293T cells by means of Lipofectamine® 2000, which injects the DNA into the nucleus of the eukaryotic cell. After an incubation period, the protein was extracted from the cell culture media. The activity of the protein was assessed using a modified Ellman assay (Ellman, Courtney, Andresjr, & Featherstone, 1960). Reactivation was measured using an Ellman assay at various time points after the reactivator, HI-6, was introduced. All agent operations were performed by the mentor. Graph 2: This graph shows the reactivation of GB-inhibited GPAChE after being exposed to HI-6. The wild type GPAChE showed no detectable reactivation and the mutant GPAChE showed reactivation of approximately 20%. References Ballantyne, B., & Marrs, T. C. (1992). Overview of the biological and clinical aspects of organophosphates and carbamates. In B. Ballantyne & T. C. Marrs (Eds.), Clinical and experimental toxicology of organophosphates and carbamates (3-14). Oxford: Butterworth-Heinemann. Cadieux, C., Broomfield, C., Kirkpatrick, M., Kazanski, M., Lenz, D., & Cerasoli, D. (2010). Comparison of human and guinea pig acetylcholinesterase sequences and rates of oxime-assisted reactivation. Chemico-Biological Interactions, 187(1-3), 229-233. doi: 10.1016/j.cbi.2010.04.020 Ellman, G., Courtney, K., Andresjr, V., & Featherstone, R. (1960). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology, 7, 88-95. The significance of the changes in activity was measured using GraphPad Prism software. A sum-of-squares F-test was used to determine the significance of the difference between the wild-type and mutant reactivation rates. In the comparison between wild-type and mutant HuAChE reactivation, the t1/2 , the time it takes for 50%